CN114590906A - 细菌漆酶在降解有机染料中的应用 - Google Patents
细菌漆酶在降解有机染料中的应用 Download PDFInfo
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- CN114590906A CN114590906A CN202011406126.0A CN202011406126A CN114590906A CN 114590906 A CN114590906 A CN 114590906A CN 202011406126 A CN202011406126 A CN 202011406126A CN 114590906 A CN114590906 A CN 114590906A
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- Prior art keywords
- cblac
- laccase
- mutant
- malachite green
- mut8
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- 239000000975 dye Substances 0.000 claims abstract description 25
- 108090000623 proteins and genes Proteins 0.000 claims description 18
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Abstract
本申请属于有机染料生物降解技术领域，具体涉及细菌漆酶在降解有机染料中的应用。本发明提供了一种细菌漆酶Cblac及其突变体Cblac‑Mut8在有机染料降解中应用。结果显示，经Cblac漆酶在pH4.0，50℃处理12h，或Cblac‑Mut8漆酶于60℃处理4h，可将孔雀石绿的降解率达98%以上，为有机染料，尤其是孔雀石绿染料的生物降解提供一种生物无害化技术路线。
Description
技术领域
本申请属于有机染料生物降解技术领域，具体涉及细菌漆酶在降解有机染料中的应用。
背景技术
孔雀石绿是一种三苯基甲烷染料，广泛应用于丝绸、羊毛、黄麻、棉花、纸张和丙烯酸工业，并作为水产养殖业的体外杀螨剂、食品添加剂和医疗消毒剂。然而，这种化合物已经成为高度争议，基于它对各种水生和陆生动物的重大风险。亚急性(0.10mg/L )和亚致死浓度(0.05mg/L)会导致降低血清钙和蛋白质水平，只是一个短期接触后，低至0.1孔雀石绿/ml仍对哺乳细胞是高毒性。此外，一些研究表明，这种染料的摄入会降低生育力，导致胎儿畸形，对人类和其他生物产生致癌作用。此外，孔雀石绿在环境上具有持久性。当孔雀石绿被释放到水体中，会导致阳光透射减少，进而降低光合活性和氧浓度的溶解，从而影响生境中的水生生物，且毒性可在鱼类生物中积累严重危害了环境和人体健康。因此，有效地从水体中去除孔雀石绿是至关重要的。
已知的物理化学吸附等技术，利用过氧化氢或臭氧氧化电解去除孔雀石绿，但是这些策略昂贵且低效率。生物处理的前景主要是由于其生态友好、相对高效和安全性能。不同组的微生物如细菌, 与直接用微生物处理相比，用纯化后酶进行的处理有许多优点，例如，它可以在不适应生物量的情况下，在较低的浓度、pH值、温度和盐度范围内操作，并且避免了长时间的孵育过程和生物吸附效应。此外，酶的降解能力可能不会受到可抑制细胞系统的有毒化合物的影响。因此，用游离酶处理是一种更简单、更有效的技术。
漆酶是一类广泛存在的酶，它能氧化多种酚类和非酚类芳香底物，同时还能还原为水，具有广泛的底物特异性和生态友好性（以空气中的分子氧为最终电子受体，仅以副产品的形式释放水），被认为是一种具有广阔应用前景的生物绿色工具。漆酶有单聚体、二聚体或四聚体糖基蛋白。漆酶主要来源是真菌漆酶和细菌漆酶，真菌漆酶一般为60-70 kDa，具有严重的糖基化修饰，最适反应温度一般为30℃-60℃， pH耐受范围在 3.5-7.0之间；而细菌漆酶具有底物谱宽，不需要糖基化修饰，热稳定性好，pH耐受性范围宽、耐碱性强等显著优点而被广泛关注。漆酶作为一种绿色生物催化剂，在处理耐污染的环境污染物和染料废水方面具有诱人的优势，由于漆酶可以催化大量酚类和非酚类化合物的氧化，同时把分子中的氧分解成水，能够加速水中有机污染物的降解和无害化。但是目前细菌源漆酶在孔雀石绿降解尚无应用。
发明内容
本发明的技术目的是提供细菌漆酶在降解有机染料中的应用，从而为有机染料，尤其是孔雀石绿染料的生物降解提供一种生物无害化技术路线。
为了实现上述技术目的，本发明采用以下技术方案：
细菌漆酶在降解有机染料中的应用，所述细菌漆酶为DUF152家族的Cblac漆酶或其突变体。
优选的，所述细菌漆酶为Cblac漆酶，该蛋白质的氨基酸序列如SEQ NO.2所示。
优选的，所述突变体为氨基酸序列SEQ NO.2改变一个或几个氨基酸残基得到的蛋白质。
进一步优选的，所述突变体为改变SEQ NO.2 第38、48、107、128、146、158、198、200、201、212、213、214、221、222、223共17个位点氨基酸残基得到的蛋白质，所述突变体命名为Cblac-Mut8，具体氨基酸序列如SEQ NO.3所示。
利用细菌漆酶降解有机染料的方法：在有机染料浓度2~200mg/L的液体中，加入Cblac漆酶或其突变体20~60U/l，30~80℃处理4~12h，即可。
优选的，所述有机染料为孔雀石绿。
进一步优选的，在孔雀石绿浓度2~100mg/L的液体中，加入Cblac漆酶或其突变体40 U/l，50~60℃处理4~12h。
本发明提供了一种细菌漆酶Cblac及其突变体Cblac-Mut8在有机染料降解中应用。结果显示，经Cblac漆酶在pH4.0，50℃处理12h，或Cblac-Mut8漆酶于60℃处理4h，可将孔雀石绿的降解率达98%以上，为有机染料，尤其是孔雀石绿染料的生物降解提供一种生物无害化技术路线。将Cblac漆酶或其突变体Cblac-Mut8脱除孔雀石绿后的处理液用于培养微生物细菌。结果显示，处理液对细菌生长无显著影响，证明该Cblac漆酶及其突变体Cblac-Mut8对有机染料具有优异的脱毒作用，具有很高的工业应用前景。
附图说明
图1 纯化Cblac漆酶（WT）及突变体Cblac-Mut8漆酶的SDS-PAGE电泳图；
图2 不同温度条件下的酶活测定；
图3 不同pH条件下的酶活测定；
图4 pH稳定性测定；
图5 热稳定性测定；
图6 不同温度条件下的Cblac和Cblac-Mut8漆酶酶动力学曲线；
图7 Cblac漆酶对孔雀石绿的脱色效果；
图8 Cblac漆酶处理不同时间的孔雀石绿降解率对比；
图9 Cblac漆酶处理液培养大肠埃希菌的生长曲线；
图10 Cblac漆酶（WT）和Cblac-Mut8（突变体）氨基酸序列比对；
图11 60℃孵育30min Cblac（WT）与突变体Cblac-Mut8酶活对比；
图12 突变体Cblac-Mut8漆酶对孔雀石绿的脱色效果；
图13 突变体Cblac-Mut8漆酶处理不同时间的孔雀石绿降解率对比；
图14 突变体Cblac-Mut8处理液培养枯草芽孢杆菌的生长曲线。
具体实施方式
以下结合具体实施例对本发明作进一步的详细描述。
本发明所用大肠埃希菌（Escherichia coil）购自中国微生物保藏中心，保藏号CICC 10305；所用枯草芽孢杆菌（Bacillus subtilis）购自中国微生物保藏中心，保藏号CICC 10275；PET28a质粒、DH5a大肠杆菌感受态细胞、BL21(DE3)大肠杆菌感受态细胞为普通市售，现保存于河南农业大学实验室中；酵母粉、胰蛋白胨均购自法国OXOID；磷酸氢二钠、磷酸二氢钠、氯化钠均购自天津大茂；孔雀石绿购自上海源叶生物有限公司。
实施例1 Cblac漆酶的表达优化
首先根据Cblac漆酶的基因序列（参考NCBI基因序列数据库，基因序列登录号：CP001393.1）进行密码子优化，优化后的基因序列如SEQ NO.1所示。优化前的基因序列属厌氧细菌表达体系，经密码子优化后，基因序列SEQ NO.1适用好氧细菌表达体系。
以上述基因序列（南京金瑞斯生物科技有限公司合成）为模板，利用PCR试剂盒进行基因扩增，所用引物对具体为：
Cblac-NdeI-F: GTGGTGGTATCGAAGGTAGGCATATGGGCTTTGTTAAAGAAAAC
Cblac-XhoI-R: ACAAGCTTGAATTCGGATCCCTCGAGCTAACGACGAACCATACGCAG
PCR条件为：98 ℃，5 min；98 ℃，20 s；56 ℃，30 s；72 ℃，2 min30 s；72 ℃，10min；4℃，99 min；进行22个循环；
扩增完成后，将克隆序列通过限制内切酶链接到pET-28a 质粒的NcoI与XhoI间，制备重组质粒PET28a-Cblac，并将重组质粒在大肠杆菌BL21(DE3)中过量表达；感受态细胞经5000 rpm，5min离心收集，于PBS (pH=7.4)高压1000 bar破碎后经50℃孵育30min，18000g，30min离心收集，然后利用Ni离子亲和柱纯化，得到Cblac漆酶。纯化Cblac蛋白的SDS-PAGE电泳图如图1所示（WT）；从图中可以看出，在30KDa位置出现电泳条带，说明获得了纯化的Cblac蛋白。
实施例2 Cblac酶活及酶活性质测定
以2,2′-azino-bis(3-ethylbenzthiazoline)-6-sulfonate (ABTS, ε420 = 38,000 M-1 cm-1) 为底物，在20mM 醋酸-醋酸钠反应缓冲液体系中进行Cblac酶性质及酶活测定；测定结果见图2~图5；结果显示Cblac漆酶在60℃的酶活最高，最适pH值为pH4.0，酶活在50℃半衰期10h左右。
测定50℃条件下Cblac的酶促动力学：在1 mM CuSO4溶液中加入Cblac漆酶10U/L，反应体系200μl，反应时间20min；通过测定420nm波长处的吸光值来确定反应速率，所得酶促动力学曲线见图6；可以看出，Cblac漆酶的Km =2.31，kcat=5.2 min-1，kcat/ Km=2.25。
以2,2′-azino-bis(3-ethylbenzthiazoline)-6-sulfonate (ABTS, ε420 = 38,000 M-1 cm-1) 为底物，在20mM 醋酸-醋酸钠反应缓冲液体系中添加不同的金属离子，添加浓度10mM；不同金属离子存在时，Cblac漆酶酶活对比见表1；
表1 不同金属离子对酶活的影响
从上表可以看出，Mn离子和Zn离子对实施例1所得Cblac漆酶活性有一定促进作用，且Cblac漆酶对Cl离子的耐受性较高，当Cl离子浓度达到1000mM时仍能保持28%的酶活性。
实施例3 Cblac漆酶对孔雀石绿降解效果的考察
配制孔雀石绿母液500mg/L；将母液加入20mM 醋酸-醋酸钠缓冲体系，使缓冲体系中孔雀石绿浓度为50mg/L；调节pH值4.0，加入Cblac漆酶40U/l，于50℃处理24h，考察Cblac漆酶对孔雀石绿染料的降解效果。
Cblac漆酶对孔雀石绿的脱色效果见图7，从图中可以看出，经Cblac漆酶在pH4.0，50℃处理后，处理液在618nm处最大吸收峰明显下降，证明孔雀石绿被降解。不同处理时间缓冲液中孔雀石绿的降解率对比结果如图8所示；可以看出孔雀石绿在12h已基本被降解完全。
将上述利用Cblac漆酶降解孔雀石绿后的处理液用以培养细菌。按照体积比1:1将处理液加入液体LB培养基中，分别接种大肠埃希菌，测定一定时间内菌体生长曲线（图9）；上述试验以仅用LB培养基培养为对照组（control），每个条件设置三个平行；
从图9可以看出用稀释的处理液培养大肠埃希菌，菌体生长曲线与对照组相似。而加入50mg/L孔雀石绿的样品组，菌体生长受到严重抑制；上述结果证明，经Cblac漆酶处理孔雀石绿后，并不产生抑制细菌生长的物质，因而能够实现对孔雀石绿的生物无害化处理。
实施例4 Cblac的酶学改造及突变体Cblac-Mut8的制备
实施例1~3结果表明，利用Cblac漆酶能够实现对孔雀石绿的生物无害化处理，且Cblac漆酶的使用量小，处理效果高。
但是根据对Cblac漆酶的研究发现，Cblac漆酶的表达大部分为包涵体，酶活性较低，热稳定性较差，为了提高Cblac漆酶热稳定性和可溶性表达，利用SWISS-MODEL结构模拟（PDB）结构提交至PROSS软件对Cblac漆酶氨基酸序列进行突变位点预测，得到突变体Cblac-Mut8漆酶序列，具体氨基酸序列如SEQ NO.3所示。预测的突变体Cblac-Mut8氨基酸序列与Cblac漆酶（WT）比对如图10，图中—代表a螺旋，→代表β折叠片，可以看出，突变体与野生型相比其突变位点共17个。
根据突变体氨基酸序列得到Cblac-Mut8漆酶的基因序列，具体如SEQ NO.4所示。以该基因序列（南京金瑞斯生物科技有限公司合成）为模板，并通过限制内切酶链接到pET-28a 质粒的NcoI与XhoI间，得到重组质粒PET28a-Cblac-Mut8；在实验室将重组质粒导入大肠杆菌DH5a中，37℃，12h后提质粒测序（北京擎科生物科技有限公司）；将质粒在大肠杆菌BL21(DE3)中过量表达，细胞经5000 rpm，5min离心收集，在PBS (pH=7.4)高压1000 bar条件下破碎后经50℃孵育30min，18000g，30min离心，用Co+亲和柱纯化，得到Cblac-Mut8纯酶。所得纯化突变体Cblac-Mut8的SDS-PAGE电泳图见图1。
实施例5 突变体Cblac-Mut8与Cblac（WT）酶活测定与对比
以2,2′-azino-bis(3-ethylbenzthiazoline)-6-sulfonate (ABTS, ε420 = 38,000 M-1 cm-1) 为底物，在20mM 醋酸-醋酸钠反应缓冲液体系中进行突变体Cblac-Mut8的酶活及酶性质测定。60℃孵育30min后，Cblac（WT）与突变体Cblac-Mut8两种粗酶活性对比见图11。可以看出60℃孵育30min，WT酶活显著降低，而突变体Cblac-Mut8活性基本无损失。
两种酶的最适温度、pH值和稳定性测定见图2~图6。从图2可以看出，两种酶均在60℃时酶活最高，但是其他温度条件下，突变体Cblac-Mut8的酶活更高。从图3可以看出，两种酶的最适pH值均为4.0。从图4和图5可以看出，与野生型WT相比，突变体Cblac-Mut8的pH稳定性更高，且对于温度的耐受力更强。
分别测定50℃和60℃条件下突变体Cblac-Mut8酶动力学：用终浓度酶10 U/L，在1mM CuSO4、不同浓度 ABTS条件下反应20min，测定酶促反应速率，用Graphpad prism 8拟合酶动力学曲线。上述酶促动力学曲线以50℃时Cblac漆酶（WT）动力学曲线为对照（图6）。可以看出，50℃时Cblac漆酶的Km =2.31，kcat=5.2 min-1；同为50 oC 时，突变体Cblac-Mut8的K m =1.46 mM，k cat = 31.2 min-1；而60 oC时Cblac-Mut8的K m =1.56 mM，k cat = 204.1min-1。经计算可得50℃条件下突变体Cblac-Mut8的催化活性（kcat/ Km）较WT提高了9.46倍。
实施例6 突变体Cblac-Mut8对孔雀石绿的降解效果
配制孔雀石绿母液500 mg/L；将母液加入20 mM 醋酸-醋酸钠缓冲体系，使缓冲体系中孔雀石绿浓度为分别为50 mg/L和100 mg/L；调节pH值4.0；加入Cblac-Mut8漆酶，添加量为40 U/l，于60℃处理4h，考察Cblac-Mut8对孔雀石绿染料的降解效果；Cblac-Mut8对孔雀石绿的脱色效果见图12。
从图中可以看出，经Cblac-Mut8在pH4.0、60 ℃处理后，618 nm处最大吸收峰明显下降，证明孔雀石绿被降解。不同处理时间的缓冲液中孔雀石绿的降解率对比见图13。可以看出，当孔雀石绿浓度为100mg/L时，Cblac-Mut8处理4 h即可对孔雀石绿的降解率达98%以上。
实施例7 突变体Cblac-Mut8降解孔雀石绿的处理液对微生物生长的影响
利用突变体Cblac-Mut8降解孔雀石绿后的处理液进行生物培养，考察处理液的无害化效果。
按照1:1将处理液加入液体LB培养基中，分别接种枯草芽孢杆菌，测定一定时间内菌体生长曲线（图14）；以不添加处理液的培养为空白组，以添加100 mg/L孔雀石绿的培养为对照组；
可以看出，添加Cblac-Mut8的处理液培养枯草芽孢杆菌，菌体生长曲线与空白组相似；而加入100mg/L 孔雀石绿的对照组菌体生长受到严重抑制。说明经Cblac-Mut8降解孔雀石绿后，并不产生抑制枯草芽孢杆菌生长的物质，证明该突变体对有机染料具有优异的脱毒作用，能够实现对孔雀石绿的生物无害化处理。
结论与分析
为了进一步揭示本发明所得突变体Cblac-Mut8对孔雀石绿的降解效果，将本申请的脱色效果与现有已知漆酶的脱色效果进行比对。已知漆酶的数据参照下列文献，具体对比结果见表2；
表2突变体Cblac-Mut8与现有已知漆酶对孔雀石绿染料降解效果对比
从上表可以看出，所得突变体Cblac-Mut8对孔雀石绿的降解能力强于大多数已报到漆酶。而且，该酶的最适温度为60℃，对热稳定性高：与Cblac漆酶（WT）相比，60℃时酶活半衰期提高至26.6h，是一种具有很高工业应用前景的漆酶。
参考文献：
1.Characterization of a Highly Thermostable and Organic Solvent-Tolerant Copper-Containing Polyphenol Oxidase with Dye-Decolorizing Abilityfrom Kurthia huakuii LAM0618 T
2.Functional expression enhancement of Bacillus pumilus CotA-laccasemutant WLF through site-directed mutagenesis, Enzyme and microbial technology109 (2018) 11-19.
3.High-level expression of a bacterial laccase, CueO from Escherichiacoli K12 in Pichia pastoris GS115 and its application on the decolorizationof synthetic dyes, Enzyme and microbial technology 103 (2017) 34-41
4.Cloning and functional analysis of a new laccase gene from Trametessp. 48424 which had the high yield of laccase and strong ability fordecolorizing different dyes, Bioresour Technol 102(3) (2011) 3126-37.
5.Malachite green decolourization and detoxification by the laccasefrom a newly isolated strain of Trametes sp, 63(5) (2009) 600-606.
6.Biodegradation, Enhanced biodegradation and detoxification ofmalachite green by Trichoderma asperellum laccase: Degradation pathway andproduct analysis. (2017) 258-268。
SEQUENCE LISTING
<110> 河南农业大学
<120> 细菌漆酶在降解有机染料中的应用
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ttgaagcact gaagattgat catcgcaaca ttttttatgc caaacaggtt cataagaacg 240
atatcattat cgttgaaaga ggttttgatt tcttcgaata taaccaggaa gttgaagccg 300
atggtctggt gaccaatgtt catggtattg cactgattac catgcatgca gattgttttc 360
ctgtgtacat tgttgataca aaaacccgtg tgattagcct gattcatagt ggatggcgtg 420
gtactctgca gcacattacc gaaaatgcac ttcagatttt aaagaaaaag ttcctgtcta 480
gcgccgagga tctgctggtg gcaattggtc cgggtatttg caaacggcat tttgaagttg 540
gtaaagatgt ttatgagatg tttctgcgtg aatttggtga tgaagtgtgt ctggaatcaa 600
aagaaagctt ttttatcgat ctgaagaagg caattatgat tgatttaaag aagaacggga 660
tcgaaagttg tcagattatt tcttgtgata tgtgtaccta tgaggatgca gatctgttct 720
tttcatatcg ccgcgatctg aatcggcctg agaagctggg ttctatggtt gcaattctgc 780
gtatggttcg tcgtctcgag 800
<210> 2
<211> 272
<212> PRT
<213> 人工合成
<400> 2
Met Gly Phe Val Lys Glu Asn Ile Asn Gly Ile Glu Ile Phe Arg Ile
1 5 10 15
Ser Glu Phe Glu Asp Tyr Gly Ile Glu Gly Phe Phe Thr Thr Arg Lys
20 25 30
Gly Cys Gly His Asp Ser Phe Asn Leu Ser Tyr Lys Trp Thr Ala Gln
35 40 45
Lys Asp Glu Val Asp Lys Asn Phe Arg Ile Leu Phe Glu Ala Leu Lys
50 55 60
Ile Asp His Arg Asn Ile Phe Tyr Ala Lys Gln Val His Lys Asn Asp
65 70 75 80
Ile Ile Ile Val Glu Arg Gly Phe Asp Phe Phe Glu Tyr Asn Gln Glu
85 90 95
Val Glu Ala Asp Gly Leu Val Thr Asn Val His Gly Ile Ala Leu Ile
100 105 110
Thr Met His Ala Asp Cys Phe Pro Val Tyr Ile Val Asp Thr Lys Thr
115 120 125
Arg Val Ile Ser Leu Ile His Ser Gly Trp Arg Gly Thr Leu Gln His
130 135 140
Ile Thr Glu Asn Ala Leu Gln Ile Leu Lys Lys Lys Phe Leu Ser Ser
145 150 155 160
Ala Glu Asp Leu Leu Val Ala Ile Gly Pro Gly Ile Cys Lys Arg His
165 170 175
Phe Glu Val Gly Lys Asp Val Tyr Glu Met Phe Leu Arg Glu Phe Gly
180 185 190
Asp Glu Val Cys Leu Glu Ser Lys Glu Ser Phe Phe Ile Asp Leu Lys
195 200 205
Lys Ala Ile Met Ile Asp Leu Lys Lys Asn Gly Ile Glu Ser Cys Gln
210 215 220
Ile Ile Ser Cys Asp Met Cys Thr Tyr Glu Asp Ala Asp Leu Phe Phe
225 230 235 240
Ser Tyr Arg Arg Asp Leu Asn Arg Pro Glu Lys Leu Gly Ser Met Val
245 250 255
Ala Ile Leu Arg Met Val Arg Arg Leu Glu His His His His His His
260 265 270
<210> 3
<211> 272
<212> PRT
<213> 人工合成
<400> 3
Met Gly Phe Val Lys Glu Asn Ile Asn Gly Ile Glu Ile Phe Arg Ile
1 5 10 15
Ser Glu Phe Glu Asp Tyr Gly Ile Glu Gly Phe Phe Thr Thr Arg Lys
20 25 30
Gly Cys Gly His Asp Asp Phe Asn Leu Ser Tyr Lys Trp Thr Ala Arg
35 40 45
Lys Asp Glu Val Asp Lys Asn Phe Arg Ile Leu Phe Glu Ala Leu Lys
50 55 60
Ile Asp His Arg Asn Ile Phe Tyr Ala Lys Gln Val His Lys Asn Asp
65 70 75 80
Ile Ile Ile Val Glu Arg Gly Phe Asp Phe Phe Glu Tyr Asn Gln Glu
85 90 95
Val Glu Ala Asp Gly Leu Val Thr Asn Val Pro Gly Ile Ala Leu Ile
100 105 110
Thr Met His Ala Asp Cys Phe Pro Val Tyr Ile Val Asp Thr Lys Asn
115 120 125
Arg Val Ile Ser Leu Ile His Ser Gly Trp Arg Gly Thr Leu Gln His
130 135 140
Ile Val Glu Asn Ala Leu Gln Ile Leu Lys Lys Lys Phe Asn Ser Ser
145 150 155 160
Ala Glu Asp Leu Leu Val Ala Ile Gly Pro Gly Ile Cys Lys Arg His
165 170 175
Phe Glu Val Gly Lys Asp Val Tyr Glu Met Phe Leu Arg Glu Phe Gly
180 185 190
Asp Glu Val Cys Leu Lys Ser Gly Gly Ser Phe Phe Ile Asp Leu Lys
195 200 205
Lys Ala Ile Glu Tyr Leu Leu Lys Lys Asn Gly Ile Lys Pro Glu Gln
210 215 220
Ile Ile Ser Cys Asp Met Cys Thr Tyr Glu Asp Glu Asp Leu Phe Phe
225 230 235 240
Ser Tyr Arg Arg Asp His Asn Arg Pro Glu Lys Leu Gly Ser Met Val
245 250 255
Ala Ile Leu Arg Met Val Arg Arg Leu Glu His His His His His His
260 265 270
<210> 4
<211> 798
<212> DNA
<213> 人工合成
<400> 4
atgggctttg taaaagaaaa tataaacggg atcgaaattt ttcgtatctc tgagttcgag 60
gactacggca tcgagggctt tttcaccacg cgtaagggtt gcggtcatga tgatttcaat 120
ctgtcctaca aatggaccgc gcgtaaggac gaagtcgata aaaatttccg catcttgttt 180
gaggcgctta agatcgacca ccgtaatatt ttttatgcga agcaggttca caaaaacgac 240
attattatcg tggagcgcgg tttcgacttc ttcgaatata accaggaggt ggaggctgac 300
ggcctggtca ccaatgttcc gggtattgcg ctgattacga tgcatgcaga ctgttttccg 360
gtttacatcg tggataccaa aaaccgtgtt atttccctga tccacagcgg ttggcgtggc 420
accctgcagc atattgtgga gaacgctttg caaattctga aaaagaagtt caacagcagc 480
gcagaagatt tgctggttgc gatcggccca ggcatctgta aacgtcactt tgaggtgggt 540
aaagatgttt atgaaatgtt tctgcgtgaa tttggcgatg aagtgtgcct gaaaagcggt 600
ggtagcttct tcatcgacct aaagaaggcc atcgagtacc tgctgaagaa gaacggtatc 660
aaaccggaac aaatcatttc gtgcgatatg tgcacttatg aggacgagga cttgttcttt 720
agctaccgca gagatcacaa ccgtccggaa aaactgggct ctatggtggc cattctccgc 780
atggttcgtc gcctcgag 798
Claims (7)
1.细菌漆酶在降解有机染料中的应用，其特征在于：所述细菌漆酶为DUF152家族的Cblac漆酶或其突变体。
2.如权利要求1所述的应用，其特征在于：所述细菌漆酶为Cblac漆酶，该蛋白质的氨基酸序列如SEQ NO.2所示。
3.如权利要求2所述的应用，其特征在于：所述突变体为氨基酸序列SEQ NO.2改变一个或几个氨基酸残基得到的漆酶。
4.如权利要求3所述的应用，其特征在于：所述突变体为改变SEQ NO.2 第38、48、107、128、146、158、198、200、201、212、213、214、221、222、223共17个位点氨基酸残基得到的蛋白质，所述突变体命名为Cblac-Mut8，具体氨基酸序列如SEQ NO.3所示。
5.如权利要求1~4任一项所述的应用，其特征在于：在有机染料浓度2~200mg/L的液体中，加入Cblac漆酶或其突变体20~60U/l，30~80℃处理4~12h，即可。
6.如权利要求1~4任一项所述的应用，其特征在于：所述有机染料为孔雀石绿。
7.如权利要求6所述的应用，其特征在于：在孔雀石绿浓度2~100mg/L的液体中，加入Cblac漆酶或其突变体40 U/l，50~60℃处理4~12h。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994524A (zh) * | 2012-11-16 | 2013-03-27 | 南京农业大学 | 一种漆酶基因及其编码的蛋白和应用 |
| CN105671011A (zh) * | 2016-03-25 | 2016-06-15 | 中国农业科学院农业资源与农业区划研究所 | 细菌类漆酶laclK的基因和蛋白及应用 |
| CN110218708A (zh) * | 2019-06-20 | 2019-09-10 | 天津科技大学 | 一种细菌漆酶及其基因、制备方法与应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994524A (zh) * | 2012-11-16 | 2013-03-27 | 南京农业大学 | 一种漆酶基因及其编码的蛋白和应用 |
| CN105671011A (zh) * | 2016-03-25 | 2016-06-15 | 中国农业科学院农业资源与农业区划研究所 | 细菌类漆酶laclK的基因和蛋白及应用 |
| CN110218708A (zh) * | 2019-06-20 | 2019-09-10 | 天津科技大学 | 一种细菌漆酶及其基因、制备方法与应用 |
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